Supercritical fluid-assisted nebulization and bubble drying

ABSTRACT

A method of making fine dry particles of substances is provided by forming a composition comprising a substance of interest and a supercritical or near critical fluid; rapidly reducing the pressure on said composition, whereby droplets are formed; and passing said droplets through a flow of heated gas. The process does not require any organic solvent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application takes priority from U.S. provisional application serialNo. 60/138,394, filed Jun. 9, 1999, which is incorporated by referencein its entirety herein.

BACKGROUND OF THE INVENTION

With advances in gene therapy and recombinant DNA technology, proteinpharmaceuticals are an important class of therapeutic drugs. Forexample, pulmonary delivery of therapeutic peptides and proteins hasreceived significant attention in recent years, for the treatment ofrespiratory illness and as an attractive alternative to injection forthe systemic delivery of macromolecules. However, the commercialproduction of protein pharmaceuticals is severely limited by chemicaland physical degradation of the proteins which can lead to biologicalinactivation (Manning, M. C. et al. (1989), “Stability of ProteinPharmaceuticals,” Pharm. Res. 6:903-918; Lai, M. C. and Topp, E. M.(1999), “Solid-State Chemical Stability of Proteins and Peptides,” J.Pharm. Sci. 88:489-500). Many of these degradation processes use waterfor hydrolysis and/or other degradation pathways. Therefore, manyprotein pharmaceuticals are prepared in the solid state as dry powdersto prolong the useable shelf life of the product and the storagestability of the product. Protein unfolding in the dried solid can leadto irreversible denaturation upon immediate rehydration and significantreduction of long term storage stability.

Supercritical fluids are substances at a temperature and pressure abovea critical temperature and pressure where the substance has a density,compressibility and viscosity intermediate between a gas and a liquid.Near-critical fluids are similar to supercritical fluids and are definedas fluids within 10% of the critical temperature and the criticalpressure. For example, since the critical temperature of CO₂ is 31.6° C.(304.6K) and the critical pressure is 1073 psi, CO₂ above 2° C. (275K)and 966 psi is near-critical. Supercritical fluids have been researchedfor their use in the production of fine powders of pharmaceuticals,however these technologies (supercritical fluid nucleation (Larson, K.A. and King, M. L. (1986), “Evaluation of Supercritical Fluid Extractionin the Pharmaceutical Industry,” Biotechnol. Prog. 2:73-82), rapidexpansion of a supercritical solution (Tom, J. W. and Debenedetti, P. G.(1991), “Precipitation of Bioerodible Microspheres and Microparticles byRapid Expansion of Supercritical Solutions,” Biotechnol. Prog.7:403-41 1) and gas antisolvent techniques (Randolph, T. W. et al.(1993), “Sub-micrometer-sized biodegradable particles of poly(L-lacticacid) via the gas antisolvent spray precipitation process,” Biotechnol.Prog. 9:429; Meyer, J. D. et al. (1998), “Preparation and in vitrocharacterization of gentamycin-impregnated biodegradable beads suitablefor treatment of osteomyelitis,” J. Pharm. Sci. 87:1149; Winters, M. A.et al. (1996), “Precipitation of proteins in supercritical carbondioxide,” J. Pharm. Sci. 85:586; Palakodaty, S. et al. (1998),“Supercritical fluid processing of materials from aqueous solutions: theapplication of SEDS to lactose as a model substance,” Pharm. Res.15:1835) require that the pharmaceutical be soluble directly in thesupercritical fluid or be precipitated by the supercritical fluid fromnonaqueous solvents such as dimethylsulfoxide. The nebulizer systemdisclosed in U.S. Pat. No. 5,639,441 (Sievers, R. E. and Karst, U.,issued Jun. 17, 1997) and divisional application 08/847,310 permits theuse of mixtures of supercritical fluids with immiscible liquids such aswater to process substances that are not soluble in the supercriticalfluid to form aerosols of vapors. Therefore, using the methods anddevices disclosed in U.S. Pat. No. 5,639,441 particles of water solubleproteins, excipients, stabilizers, bulking agents and/or surfactants maybe formed rather than just particles of those compounds that are solublein supercritical fluids and/or organic solvents. U.S. Pat. No. 5,639,441is hereby incorporated by reference, to the extent not inconsistent withthe disclosure herein. Unlike the other precipitation methods, e.g., theSEDS process and GAS processes referred to above, no organic solventsare required in the new process; only the drug, water and thesupercritical or near-critical fluid (for example, carbon dioxide) areneeded.

Even though particles of water soluble proteins and other aqueousformulations can be prepared, no method to form suitable dry powders ofthese proteins and/or formulations existed until now. Existingtechnologies to produce dry protein powders, such as spray-drying,freeze-drying, or ultrasonic nebulization, suffer from a variety ofproblems. In general, dry protein powders are often irrevocablyinactivated when produced by prior art methods because the processingsteps involved in these methods, temperature required to dry theproteins using these methods and dehydration processes of these methodsdamage the delicate structure of the protein. Also, for use in directinhalation applications, powders must be small enough to allow foreffective pulmonary delivery. Drug delivery via a pulmonary route ispreferred over other delivery routes such as injections for reasons suchas decreased pain and delivery of the drug to the desired location morequickly. If the particles produced by the drying process are larger thandesired, they must be jet-milled or mechanically ground. This creates anadditional physical stress on the molecules and may impart a furtherloss of protein activity. Dry powders produced in the correct sizeregion could be used directly in dry powder inhalers for pulmonarydelivery.

Spray-drying is a currently-available method to produce dry proteinpowders. In the spray-drying technique, a jet nebulizer is used to forma plume of droplets. In one type of nebulizer, a liquid sample is suckedthrough a small diameter tube by a high-pressure stream of gas. The gasbreaks up the liquid into fine droplets. The gas can also flow acrossthe small diameter tube at right angles and form droplets in a similarmanner. Ultrasonic nebulizers use ultrasonic vibrations coupled to thesample solution that cause the solution to break up into small droplets.One disadvantage of the method of spray-drying is the plume of moleculesexiting the jet nebulizer is not very dense. This results in a processthat is slow in producing a desired amount of protein. Freeze-drying isanother currently used method to produce dry protein powders whereinaqueous solutions of drugs are frozen and placed under a vacuum tosublime the water. One disadvantage of the method of freeze-drying isthe drying process is very slow. Also, the particles produced arerelatively large, requiring additional processing steps to producepharmaceutically desirable sizes.

U.S. Pat. No. 6,063,138 (Hanna, et al., issued May 16, 2000) and relatedEP 0767702 describes methods of forming particles of a substance byco-introducing a supercritical fluid; a solution or suspension of thesubstance in a first vehicle; and a second vehicle which issubstantially miscible with the first vehicle and substantially solublein the supercritical fluid into a particle formation vessel which ismaintained at supercritical pressure and temperature.

PCT published application PCT/US99/19306 (WO 010541) (Edwards et al.)describes methods of forming particles by combining a bioactive agent, aphospholipid and an organic solvent or organic-aqueous co-solvent toform a mixture which is then spray-dried.

U.S. Pat. No. 5,695,741 (Schutt et al., issued Dec. 9, 1997) and relatedU.S. Pat. No. 5,639,443 (Schutt et al., issued Jun. 17, 1997) and U.S.Pat. No. 5,720,938 (Schutt et al., issued Feb. 24, 1998) describe“microbubbles” useful for magnetic resonance imaging and ultrasoundimaging. The “microbubbles” are prepared by spray-drying a liquidformulation to produce microspheres having voids and then permeating themicrospheres with a fluorocarbon gas osmotic agent.

U.S. Pat. No. 5,928,469 (Franks et al., issued Jul. 27, 1999) describesmixing materials with a carrier substance that is water-soluble orwater-swellable and spray drying the resultant mixture to form particlescontaining both the material and the carrier substance in which thecarrier substance is in an amorphous (glassy or rubbery) state. Franksdescribes spray drying at gas temperatures of 100 to 300° C.

U.S. Pat. No. 6,001,336 (Gordon et al., issued Dec. 14, 1999) describesspray drying suspensions of a hydrophobic component and a hydrophiliccomponent dissolved in an aqueous solution.

U.S. Pat. No. 5,851,453 (Hanna et al., issued Dec. 22, 1998) and relatedEP 0 706 421 describe a method and an apparatus for forming particulateproducts by introducing a supercritical fluid and a solution orsuspension of a substance in a vehicle soluble in the supercriticalfluid into a vessel which is maintained at controlled temperature andpressure. WO 95/01324 (York et al., published Jan. 12, 1995) describesparticles of salmeterol xinafoate using this method.

WO 99/16419 (Tarara et al., published Apr. 8, 1999) describes preparing“perforated microstructures” by atomizing a liquid and spray drying theliquid droplets that are formed. WO 00/00215 (Bot et al., published Jan.6, 2000) describes delivery systems of “perforated microstructures”containing “bioactive agents”.

WO 99/59710 (Hanna et al., published Nov. 25, 1999) describes a methodand apparatus for forming particles of a substance by dissolving orsuspending the substance in a first vehicle which is or contains a firstsupercritical or near critical fluid and passing that solution orsuspension into a particle formation vessel which contains a secondsupercritical fluid. The vessel is maintained at temperatures andpressures so that the second fluid remains supercritical.

WO 98/36825 (Hanna et al., published Aug. 27, 1998) describes a methodand apparatus for forming particles by directing two supercriticalfluids, one containing the substance of interest, into a heated andpressurized chamber.

There is a need for stable or pharmaceutically-active proteins in dryform, and a method to produce stable or pharmaceutically-active proteinsin dry form. Also, there is a need to produce smaller particles withimproved pharmaceutical activities.

BRIEF SUMMARY OF THE INVENTION

This invention provides a method for forming fine dry particlescomprising:

(a) forming a composition comprising one or more substances and asupercritical or near critical fluid;

(b) rapidly reducing the pressure on said composition, whereby dropletsare formed;

(c) passing said droplets through a flow of gas heated from about 2° C.to about 300° C.

Preferably bubble drying should be conducted at temperatures aboveambient temperature and below 100° C. to minimize degradation of thepharmaceuticals. Given sufficient residence time and dilution, a flow ofdry gas will dry the fine droplets without external heating. Heatingaccelerates drying by increasing the vapor pressure of water. Thecomposition may also comprise an aqueous solvent.

Also provided is a method of forming fine dry particles comprising:

(a) mixing an aqueous solution containing the substance of interest anda supercritical or near supercritical fluid, forming a composition;

(b) rapidly reducing the pressure on said composition, whereby dropletsare formed;

(c) passing said droplets through a flow of gas heated from about 2° C.to about 300° C.

Also provided is a method of forming fine dry particles comprising:

(a) equilibrating an aqueous solution of the substance of interest witha supercritical or near supercritical fluid, forming a composition;

(b) rapidly reducing the pressure on said composition, whereby dropletsare formed;

(c) passing said droplets through a flow of gas heated from about 2° C.to about 300° C.

Also provided is a device for forming fine dry particles, consistingessentially of:

(a) a first pressurized chamber containing a first nongaseoussupercritical or near critical fluid;

(b) a second chamber containing a solution or suspension of a substancein a second nongaseous fluid;

(c) a mixing chamber for mixing said solution or suspension and firstfluid connected to said first and second chambers by conduits;

(d) first flow control means connected to the conduit between the firstchamber and the mixing chamber for passing said first fluid into saidmixing chamber;

(e) second flow control means connected to the conduit between thesecond chamber and the mixing chamber for passing said second fluid intosaid mixing chamber;

(f) a restrictor connected to said mixing chamber for conducting thecomposition out of the mixing chamber into a rapid expansion regionhaving a pressure below that of the supercritical or near critical fluidwhere a dispersion of fine particles of said substance is formed;

(g) a drying chamber connected to the restrictor;

(h) a source of gas connected to the drying chamber at one or moreinlets;

(i) means for collecting particles after they pass through the dryingchamber.

The mixing chamber is preferably a low dead volume tee.

Fine particles are those with diameter less than about 5 micrometers.Particles formed by the methods of the invention may vary in diameterbetween about 0.1 micron to about 5 microns. The particles produced maybe smaller than 0.1 microns, but current detection methods are sizelimited in the lower size range, and small particles do not constitute asignificant fraction of the mass. The particles may be of anydistribution of diameters. For certain applications, for exampleinhalation therapy, it is preferred that most particles be within therespirable range for delivery to the deep lung alveoli. Preferably theparticles range in size from 1 to 3 microns for inhalation applications.Particles may be different sizes for other applications, as known to theart or readily determined without undue experimentation. It is preferredthat for inhalation applications, the particles have a small variancefrom the average size.

As used herein, “dry” or “dried” include particles that include somemoisture, preferably not more than 5% by weight Dry particles includethose particles which include from 0.0001% to 1% moisture, from 1% to 3%moisture, from 1% to 5% moisture, from 5% to 10% moisture, andcombinations of those ranges.

Particles of various shapes are included within the invention. Forexample, particles may be hollow, or “bubbles”. Bubbles arehollow-centered, similar to a tennis ball or a ping pong ball, althoughthey may not be as spherical as a tennis ball or a ping pong ball. Thediameter of the particle is typically about 10 to 10,000 times thethickness of the skin. Other particles formed by the method of theinvention are not hollow. Higher drying temperatures (˜100° C.) favorsforming hollow particles, while the same substance may give solidspheres if dried slower at lower temperatures.

“Composition” does not mean all substances are necessarily soluble ineach other. Substances which may be made into particles by the methodsof the invention include any substance which is either soluble in asupercritical fluid or near critical fluid or mixtures thereof; or asubstance which is soluble or suspendable in an aqueous solution. Theaqueous solution may also include various co-solvents, but avoiding theuse of organic solvents may have environmental and toxicologicalbenefits. Some substances which may be made into particles include: aphysiologically active composition comprising one or more substancesselected from the group consisting of surfactants, insulin, amino acids,enzymes, analgesics, anti-cancer agents, antimicrobial agents, viruses,antiviral agents, antifungal pharmaceuticals, antibiotics, nucleotides,DNAs, antisense cDNAs, RNAs, peptides, proteins, immune suppressants,thrombolytics, anticoagulants, central nervous system stimulants,decongestants, diuretic vasodilators, antipsychotics, neurotransmitters,sedatives, hormones, anesthetics, anti-inflammatories, antioxidants,antihistamines, vitamins, minerals and other physiologically activematerials known to the art. Particles of monoclonal antibodies andvaccines may be produced. Particles of substances such as sodiumchloride may also be produced. Some substances may bepharmaceutically-active. “Pharmaceutically-active protein” indicatesthat the protein has sufficient activity so as to be pharmaceuticallyuseful. Other substances may not be physiologically orpharmaceutically-active.

Various additives may be used in the methods and particles of theinvention. These additives may be added to the substance of interest orany solvent used in the process. Additives may also be added directly tothe particles after formation. Additives include stabilizers,excipients, bulking agents and surfactants. The use of stabilizers inprotein formulations protects against loss of protein activity upondrying. Stabilizers include, without limitation, sugars and hydrophilicpolymers, such as polyethylene glycol, hydroxy ethyl starch, dextran orothers. The stabilizer is preferably a sugar or mixture of differentsugars. Sugars that can be used include mannitol, sucrose, lactose andtrehalose, and other mono-, di-, and oligosaccharides. Addition of oneor more stabilizers to the protein solution prior to dehydrationsignificantly inhibits the conformational changes within the proteinthat are believed to be linked to a loss of enzymatic activity. One ormore surfactants can be added to alleviate stresses between droplet/airinterfaces and decrease degradation that may occur upon drying.Surfactants may be added to alleviate agglomeration or clumping that mayoccur upon drying. Surfactants are also thought to assist in theformation of spherical particles. Examples of surfactants that may beused include: polyoxyethylene (20) sorbitan surfactants (Tweens), suchas Tween 20, Tween 40, Tween 80 and Tween 85; stearic acid; myrj (PEGmonostearates), Span 85 (sorbitan trioleate) and polyether-carbonateblock copolymers of the type reported in Beckman et al. (2000) Nature405:165. Phospholipids, including phosophoglyceride may be used.Surfactants and other agents, such as guanidinehydrochloride, mayfacilitate protein refolding, coupled with pressure treatment. (St.John, R. J. et al. (1999) Proc. Natl. Acad. Sci. 96:13029). Bulkingagents and excipients may be inert or active.

If needed for stability of the final formulation, buffer is preferablyadded first. If more stability is required, sugar may be added. If stillmore stability may be added, surfactant may be added. A pH bufferingsubstance is useful to counteract the rapid drop in pH from carbondioxide dissolution at high pressure to form carbonic acid.

For low potency drugs, the fraction of additives in the powder should beminimized; for high potency drugs, inactive excipients (diluents)sometimes constitute more than 99% of the mass. Additives may be addedin any useful amount to the composition. Additives are typically used ata concentration of between about 0.001 to 0.5 wt % of surfactant(preferably between about 0.001 to 0.1 wt %), and between about 0.05 to25% of stabilizer (preferably, when sugar is used, between about 0.1 to20%, limited by the solubility limit of sugar) by weight to a solutioncomprising a protein of interest. These percentages are expressed forthe aqueous solution before spraying and drying. The final percentage ofsugar in the dried powder can be as high as ˜99.9%.

The composition may comprise a substance which is soluble in thesupercritical or near critical fluid, such as a lipophilic compound. Amixture of supercritical or near critical fluids may be used. Thecomposition may also comprise an aqueous solution or suspension of thesubstance and a supercritical or near critical fluid (or fluids).Droplets of substances which are not soluble in the supercritical ornear critical fluid, such as hydrophilic substances, are then formed. Inone embodiment of this method, an aqueous solution comprising adissolved or suspended compound is pumped into a low-dead volume teewhile a supercritical or near critical fluid is pumped into another legof the tee. The resulting emulsion or supercritical solution pressurizedto a pressure near or above the critical pressure of the supercriticalfluid (about 70 to about 100 atmospheres when carbon dioxide is used) isallowed to expand to atmospheric pressure out a flow restrictor, formingfine droplets or bubbles containing the dissolved drug species. Thisaerosol is directed into a drying chamber where solvent evaporation andparticle formation takes place.

A second variant of this method allows aerosols to be formed without alow-dead volume tee. In this method, an aqueous solution of precursorsis first allowed to equilibrate with a near critical or supercriticalfluid in a static canister or chamber, preferably with stirring oragitation. This composition is then allowed to expand to atmosphericpressure out of a flow restrictor, forming fine droplets. These dropletsare directed into a drying chamber where solvent evaporation andparticle formation takes place.

The initial concentration of the substance of interest in either thesupercritical fluid or the aqueous solvent (or solvent mixture) islimited only by the solubility or saturation point of the substance inthe solvent or supercritical fluid. Typical starting concentrations areabout 1% to about 25% w/w of total solids in the solution or fluid.

Preferably the supercritical fluid is carbon dioxide because carbondioxide is endogenous and relatively non-toxic, as well as having acritical pressure and temperature easily obtainable. Other supercriticalor near-critical fluids may be used, provided that the criticaltemperature and pressure are obtainable and useful. Carbon dioxide iscurrently less expensive than any organic solvent and its use avoids VOCemissions. Carbon dioxide's solubility in water is about 2% at 100 atmat near-ambient temperatures.

A number of fluids suitable for use as supercritical fluids are known tothe art, including carbon dioxide, sulphur hexafluoride,chlorofluorocarbons, fluorocarbons, nitrous oxide, xenon, propane,n-pentane, ethanol, nitrogen, water, other fluids known to the art, andmixtures thereof. The supercritical fluid is preferably carbon dioxideor mixtures or carbon dioxide with another gas such as fluoroform orethanol. Carbon dioxide has a critical temperature of 31.3 degrees C.and a critical pressure of 72.9 atmospheres (1072 psi), low chemicalreactivity, physiological safety, and relatively low cost. Anotherpreferred supercritical fluid is nitrogen.

The gas that contacts the flow of the droplets is preferably inert, anda preferred embodiment is nitrogen gas, but the gas may be chosen so asto react with the molecule of interest in the course of drying.Preferably the flow of gas contacting the flow of the sample forms asheath surrounding the flow of the sample, but the flow of gas maycontact the flow of the sample by other means such as turbulent mixing.Preferably this gas is heated to a temperature sufficient to cause thedesired level of particle drying and also not substantially degrade thebiological activity of particles. The gas may be heated to between about2° C. to about 300° C., preferably below 100° C., although depending onthe substance being dried and the constituents of the composition, thetemperatures may be adjusted. A preferred range of drying temperaturesis between about 35° C. to about 100° C.

Preferably the gas is contained in a drying chamber such as a dryingtube. The drying tube is as long as necessary to produce particleshaving the desired level of moisture by the time the particles reach theend. The drying tube is preferably larger than the diameter of thedroplet plume formed from the rapid expansion. The drying tube may beheated, but that is not required. Preferably most of the heat requiredfor vaporizing water is provided by heating the drying gas before itenters the drying chamber. Heating the drying tube may assist inpreventing condensation on the surfaces of the tube. The drying tube maybe heated externally by means of a lamp such as an infrared lamp, orinternally by any means known in the art such as a heating wire imbeddedin the material making up the tube. The drying tube may be made from anysuitable material which can withstand the temperatures to which it issubjected. Examples of material from which the drying tube can be madeare stainless steel or borosilicate glass. Any other design of dryingchamber may be used if the desired results are obtained. Otherapparatuses, for example a microwave oven, may be used in place of thedrying tube to perform the same function.

Rapid reduction of the pressure of the composition is typicallyperformed using a flow/pressure restrictor. The restrictor may be ahollow needle of an thermally conductive material such as stainlesssteel or a ceramic material, or other material which is able towithstand the pressure and temperature placed upon it. The restrictormay alternatively be a fused silica flow restrictor, or a ceramicmulti-channel bundle of capillaries such as that discussed in moredetail elsewhere. Also, high pressure sintered stainless steel filtersmay be used to generate aerosols. The length of the restrictor istypically about 2 inches long; however, the length must not be so longas to cause low flow rates or the precipitation of sufficient solidsubstance in the restrictor to cause clogging. The restrictor may haveas large a diameter as desired, as long as the desired size particlesare formed and the pumps have sufficient capacity to maintain thepressure. The lower limit of diameter is determined by the viscosity ofthe solution being passed through the restrictor. If the viscosity istoo high, particles are not formed.

The invention also provides a multichannel restrictor. These openingsmay be spaced approximately the same distance from each other. Thestructure may have a cylindrical, a hexagonal, or other shape whichallows it to be coupled with the other components used. The openings maybe any suitable shape, such as round or hexagonal. An embodiment of onesuch structure has about 900 non-concentric parallel channels in about a2 mm total diameter. This multichannel restrictor may have a totaldiameter which provides the desired particle formation. Preferably eachchannel has an inner diameter between about 40 μm and about 125 μm.Other multichannel structures may be used, and the openings do not needto be a similar size, although it is preferred if the openings aresimilarly sized.

The exit tip of the restrictor structure may be flat or substantiallyflat, or may be shaped. One shape that is particularly useful is formedby removing material from the sides of a flat end, to form an elongatedpoint, similar to a pencil. This modification gives a more dispersedstream of droplets emitted over a 180° angle which is useful to assistin preventing agglomeration of particles as they undergo bubble drying.The particular geometry which gives the best results, depending on theresults desired, may be discovered by routine experimentation. Any othermeans available for reducing the pressure on a composition may be usedto accelerate drying.

With many openings through which the composition may pass, the flow ratethrough the system may be increased and the throughput of the systemincreased. The overall throughput rate is principally controlled by thetotal inner diameter of the flow restrictor. Various inner diameters ofsingle channel restrictors may also be used, including 75 micron, 100micron, 170 micron, and 200 to 1000 micron. Another benefit ofmulti-channel restrictors over single channel ones is that if onechannel becomes clogged, the remainder of the channels remainfunctional.

The method of this invention may be used for processes in which fasterdrying than currently available is desired. Fast drying may occurbecause the swelling/bursting processes of the invention gives greatersurface area for drying, although applicants do not wish to be bound bythis theory.

Various particles including pharmaceutically-active protein compositionsin dry form comprising particles of a protein of interest and optionallycontaining one or more additives selected from the group consisting ofexcipients, stabilizers, bulking agents and surfactants, wherein theadditives are present at a concentration of about 0.001% to about 99.9%,measured by weight of the dry protein, and wherein the particles havediameters of about 0.1 microns to about 10 microns are produced by themethod of this invention. Particles may have a variety of bulk densitiesdepending on the particular substances involved and the conditions underwhich particle formation and drying occur. For example, particles withbulk densities of between about 0.1 and 1.5 g/cm³ may be formed. Thebulk density may be less than 1 g/cm³, less than 0.8 g/cm³, less than0.5 g/cm³, less than 0.4 g/cm³ or other ranges. Particles may have avariety of activities after rehydration. For example, dry particles withat least 90% of the original activity upon rehydration are included inthe invention. Particles with 90-95%, 90-100%, 100-120% originalactivity are also among those included in the invention.

The particles may be stored in any convenient manner after formation anddrying, including placing in bags or other storage devices or additionaldrying during storage over desiccants such as P₄O₁₀ can be undertaken.

The invention also provides a nebulization system using an injectionport that requires a lower volume of sample than currently availablesystems. This is an advantage when conducting laboratory scaleexperiments, for example, on costly samples. The injection port alsopermits equilibration of the nebulization and drying system with thesolvents only, followed by introduction of a solution comprising theprotein of interest after the system has been equilibrated. This reduceswaste of the protein. This nebulization system may be attached to abubble drying system, or may be coupled with equipment used inconventional spray drying to permit faster drying at lower temperatures.

A method of making droplets by injecting a volume of one or moresubstances into a flow of a solution comprising an aqueous orsupercritical or near supercritical solution using a means forintroducing a small sample volume into the flow and subjecting theresulting solution to a rapid pressure decrease to form droplets isprovided. Preferably the introducing means is an injection port, such asthat used for HPLC. Preferably the volume introduced is about 0.1 mL toabout 10 mL.

A method of delivery of proteins or other substances comprising: dryinga protein or other substance using the method of this invention,reconstituting the protein with water or other suitable substance, anddelivering via desired means is provided.

Stable and/or pharmaceutically active particles are also provided.“Stable” means resistant to decomposition during storage, shipping,reconstitution, and administration.

A device for rapid expansion of a composition comprising a low deadvolume tee through which said composition passes and a restrictor withmore than one substantially parallel non-concentric channels affixed tosaid tee is also provided. A low dead volume tee is a mixing tee havinga volume of about 0.2 to 10 μl. The tee may be affixed to the restrictorby any suitable means, for example, epoxy or appropriate fittings.

The drying technique of the invention has advantages over conventionaldrying techniques. The drying technique of the invention is scalablewithout significant alteration of particle size or morphology. Themethod also provides particles having lower density than particlesproduced by other methods. This leads to particles with smallaerodynamic sizes, but large absolute particle dimensions, since in oneembodiment, additional gaseous supercritical fluid is formed and leavesthe substance of interest containing particles with porous or hollowstructures. This permits larger particles with lower momentum to reachthe deep lung than would otherwise be possible, in one application.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is a schematic diagram of a supercritical fluid assistednebulization and bubble drying system.

FIG. 2 is a diagram of a drying tube used in the invention.

FIG. 3 is an electron micrograph of the end of a multi-channel flowrestrictor.

FIG. 4 is an electron micrograph of a shaped multi-channel flowrestrictor.

FIG. 5 is a graph of the buffering capacity of monobasic/dibasicpotassium phosphate (♦, pH7), Tris/Tris HCl (▾, pH 7.2), citricacid/sodium citrate (▪, pH 5.5), and acetic acid/sodium acetate (, pH5)upon nebulization with supercritical CO₂.

FIGS. 6A-6G are electron micrographs of dry protein powders produced bythe method of this invention.

FIG. 7 shows electron micrographs of lysozyme powders produced by themethod of this invention with “Buffer only” powder (A) 10% mannitol (B),10% sucrose (C), and 10% sucrose with 0.01% Tween 20 (D).

FIGS. 8A-B shows X-ray diffraction patterns for samples prepared bysupercritical CO₂-assisted nebulization of 4 mg/mL lysozyme, 100 mMphosphate buffer at pH 7.0 and 10% mannitol (A) or 10% sucrose (B), withfinal approximate solid weight percentages in the bubble dried powdersof 4% protein, 16% buffer and 80% sugar.

FIGS. 9A-E shows FTIR spectra of lysozyme powders produced by the methodof this invention with (A) lysozyme in the native aqueous state; (B)lysozyme containing 10% sucrose and 001% Tween 20; (C) lysozyme with 10%sucrose; (D) lysozyme with 10% mannitol; and (E) lysozyme with bufferonly dried at 70° C.

FIG.10 is a graph of the lysozyme enzymatic activity of rehydratedpowders, produced using supercritical CO₂-assisted nebulization andbubble drying, compared to the activity of the original formulations.

FIG. 11 is a graph of the activity of lactate dehydrogenase (LDH)powders produced by the method of this invention after rehydration.

FIG. 12 is a graph of the recovery of LDH activity using differentexcipients upon rehydration using different percentages of excipients.

DETAILED DESCRIPTION OF THE INVENTION

The advantages of the present invention over currently used techniquesto form dried proteins include the ability to form stable formulationsof a variety of different proteins and other materials that arecurrently unsuitable for drying using presently-available techniques.The present invention imparts lower stress and less severe damage to thesubstance during drying. Also, the fine powders of stable formulationsincluding proteins formed by this method are prepared directly in theinhalable size region, eliminating the need for additional mechanicalmicronization which could impart additional stresses to the driedformulation and further loss of activity. The temperatures preferablyused in the process of the current invention are much lower than that ofconventional drying processes. This decreased temperature of dryingresults in less extensive thermal degradation. The powders formed by themethod of the current invention may retain less water in the preparedpowder than with conventional methods, eliminating the need for anadditional drying step. The present invention reduces the amount ofdenaturation and resulting aggregation that is seen with conventionalnebulization.

One specifically exemplified embodiment of the present invention isshown in FIG. 1 illustrating the flow of fluids, solutions and particlesin the method of this invention. A carbon dioxide reservoir 10 (whichcan be a gas cylinder or other reservoir) containing liquid carbondioxide is connected to a carbon dioxide pump 15 (which can be a syringepump or other suitable pump) via conduit 70. The carbon dioxide pump 15is connected to mixing tee 20 by means of conduit 71, through which thecarbon dioxide is pumped under conditions at which it becomes asupercritical fluid or near critical fluid, when it reaches the heatedmixing tee 20. Aqueous solvent reservoir 40 is connected via conduit 72to fluid pump 35, preferably a high performance liquid chromatography(HPLC) pump, which is connected via conduit 73 to the injection port 30.The injection port 30 is used to add an aqueous solution of protein orother drug, buffer, bulking agent, excipient, stabilizer and/orsurfactant. The solution of aqueous solvent from reservoir 40, andprotein solution, with or without additives injected through injectionport 30, is connected via conduit 74 to mixing tee 20. Mixing tee 20preferably has a low dead volume, e.g., less than about 10 μl so than anintimate mixture of the supercritical carbon dioxide and the aqueoussolution may be formed therein. Mixing tee 20 is equipped with pressurerestrictor 25 to maintain back-pressure in mixing tee 20, and may beoptionally equipped with heating coils to maintain supercriticaltemperature therein. Upon passage of the resulting mixture from mixingtee 20 through small diameter pressure restrictor 25, sudden release ofthe pressure at the exit of the orifice of pressure restrictor 25 occursand a very fine aerosol of solution droplets and/or bubbles is formed(80). The restrictor may comprise a single outlet, or may consist ofmany outlets. Aerosol 80 is then directed into the center of drying tube45. Drying tube 45 is preferably less than 1 meter long and 10 cm indiameter. Nitrogen gas from nitrogen reservoir 30 is added to dryingtube 45 via conduit 85 through input ports 35. Preferably a plurality ofinput ports 35 are arranged equidistant around the center of drying tube45. Lamp 90 may be used to heat drying tube 45. Nitrogen gas may also beheated as shown in FIG. 2. Heated air or mixtures of gases may be usedif there is no explosion hazard. Heat aids in the drying process andkeeps water from condensing on the inner walls of the tube and on thefilter paper. If the water condenses on the filter paper, it may causeclogging. The output of drying tube 45 is fitted with filter paperholder 50 which is connected to vacuum pump 55 through a suitableconnector 91.

In operation, the liquid carbon dioxide is pumped by means ofsupercritical carbon dioxide pump 15 from carbon dioxide reservoir 10via conduit 70 through pump 15 and via conduit 71 to the low volume (0.2to 10 μl) mixing tee 20 where it becomes a supercritical fluid (if it isnot already), or a near critical fluid. Pump 35 pumps aqueous solventfrom solvent reservoir 40 via conduit 72 where it is pumped via conduit73 to injection port 30, where the protein of interest is added as anaqueous solution. Additives may also be introduced, either throughinjection port 30 or to the solvent in reservoir 40. Buffers may be usedin the protein solution or aqueous solvent in reservoir 40 to reduce theeffect of the possibility of denaturation of the protein and reduce theproduction of aggregates that may occur due to the otherwiseuncompensated pH change due to the introduction of carbon dioxide in themixture. Mixing tee 20 may be heated by the use of heater coils, and/orrestrictor 25 may be heated to maintain the temperature above thecritical temperature of the carbon dioxide. The flow of carbon dioxideand aqueous solution are adjusted independently by means of valves, notshown in FIG.1. Flow rates can also be controlled by altering pumpingconditions. The mixture in mixing tee 20 expands downstream and formsaerosol 80 comprising fine particles of the substance dissolved orsuspended in the aqueous solution. The particles are directed in thecenter of drying tube 45 where they are dried and collected on filterpaper in filter paper holder 50. Alternatively, particles may becollected using a cyclone separator or cascade impactor, for example, ifthe particle size distribution is suitable.

The use of injection port 30 allows equilibration of the system prior tointroduction of protein and/or additives. Equilibration of the system isreached when the temperature of the drying tube is equilibrated asmeasured by a thermocouple and the flow rates of aqueous solution andcarbon dioxide reach steady states. After equilibration, a known volumeof the protein formulation into the aqueous feed line may be injectedthrough injection port 30.

A six-port injection valve may be adapted for use in a nebulizationsystem so that in the load stage, there are two separate solution loops.Solvent is pumped from an HPLC pump through the mixing tee in one loopand the sample is contained within a sample loop, for example a 3 mlsample loop, that is not connected with the solvent loop in the loadstage. During the inject stage, solvent is pumped from the HPLC pumpthrough the sample loop and to the mixing tee. This modification has theadvantage of not pumping protein or other substances of interest throughHPLC pistons and allows the ability to inject very small samples. Theamount of protein required per run and the overall run times aresubstantially decreased.

When a sample injection valve is used, typical system parametersinclude: carbon dioxide pressure 100 atm; carbon dioxide flow rate 0.3ml/min; aqueous flow rate 0.3 ml/min, using a 5 cm long fused silicarestrictor with 50 μm inner diameter.

FIG. 2 shows a more detailed view of the drying tube and drying process.The solution from mixing tee 20 is passed through restrictor tip 70 intodrying tube 45 through inlet 100, where droplets are formed. Nitrogen(or other gas, either inert, or containing a substance that is desiredto react with the aerosol particles) from reservoir 30 is passed viaconduit 85 to gas drying column 60. The gas is then passed via conduit95 to heating coil 65. In a preferred embodiment, the gas is heated toaround 70° C. and is present at a flow rate of around 15 L/min. Heatedgas is passed through conduit 100 to drying tube 45 via gas inlets 35.In a preferred embodiment, drying tube 45 is a glass tube with 5 inputports, one in the center where the aerosol is added, and 4 other inletsarranged around the center port. Lamp 90 is also used to heat dryingtube 45 externally. At the output of drying tube 45, the particles arecollected on filter paper held in holder 50.

Other configurations of drying tube 45 may be used. For example, more orfewer inlet ports 35 may be used. The gas may also be added to thecenter of the tube, and the particles added around the gas. Gas andparticles may also be mixed together in the drying tube. The interactionof the drying gas and particles in the tube affect the characteristicsof the final product.

Preferably all high pressure parts are made from stainless steel. Otherinert materials or coatings may be used. Preferably filter paper holder50 is made from stainless steel. The restrictor length is preferablyabout 2 in. (5 cm). The flow rates for the aqueous solutions in theabove apparatus are about 0.5 ml/min to about 3 ml/min. If desired, theprocess may be conducted on a larger scale or smaller scale by adjustingdimensions and flow rates while maintaining similar temperatures andpressures. In the specifically exemplified embodiment, flow rates ofcarbon dioxide and aqueous solvent are approximately 0.3 mL/min. whenusing a 50 μm inner diameter flow restrictor.

Injection port 30 allows several small volume aliquots (about 0.1 toabout 10 ml) of protein formulations to be introduced in the same amountof time as required by one large volume (>10 ml) in a system without theinjection port. This permits several protein formulations with varyingcomponents and concentrations to be dried quickly with small amounts ofprotein used in each run. This is a significant advantage to commerciallaboratory scale spray driers which require up to 100 ml of proteinsolution per experiment. However, the method of the invention may alsobe practiced with larger volumes of proteins and with larger volumes ofany substance desiring to be dried.

The restrictor may be a multichannel restrictor having a plurality ofparallel tubes. One such multichannel restrictor may be fabricated froma glass multi-channel column (Alltech, Inc., Illinois). This column maybe purchased in lengths up to 1 meter with about 900 holes through thelength of the column each hole having an inner diameter approximately 40or 50 microns or greater. A piece of such column can be used as amultichannel restrictor. One such product has a hexagonal shape (Alltechpart number 17059). Other products have a round shape where the hexagonis placed in a circle of the same diameter. An electron micrograph ofthe end of such a restrictor is shown in FIG. 3. The round shaperestrictors are easier to attach to pumps and canisters with ferruleseals and Swagelok fittings and they can also be attached to stainlesssteel tubing or tee by applying epoxy pre-polymer mixture to theexterior of the glass or ceramic tubing and slipping it into a slightlylarger steel tubing. The hexagonal voids can be filled with polymers orepoxys. These multichannel restrictors may be used in both static ordynamic processes. In a dynamic process, a low dead volume tee is usedto intimately mix streams of liquid (near critical ) or supercriticalfluid such as carbon dioxide with an aqueous solution or suspension tobe nebulized. In the static process, a solution of a substance ofinterest is pressurized by supercritical or near critical fluid at atemperature near its critical temperature. This allows some of thesupercritical or near critical fluid (up to about 1 to 2 mole percent ifcarbon dioxide is used) to be dissolved in the water. When the solutionapproaches equilibrium, with mixing, if the aqueous solution andsupercritical or near critical fluid are allowed to be ejected through apressure restrictor, an aerosol is formed as the fluid exiting thepressurized fluid returns to atmospheric pressure or lower. Themultichannel restrictor can also be used in the gas antisolvent methods,and any other methods where particles are desired.

The restrictor may have a shaped or substantially flat end. The end of arestrictor may be mechanically shaped. For example, part of the materialmay be removed using an abrasive. Some suitable abrasives includediamond-embedded nickel alloy pads, of the kind used by marble sculptorsmade by 3M, for example. Very small (micron sized) diamond crystalsabrade away the walls around each channel. The restrictor type shown inFIG. 3 after shaping with a diamond pad is shown in FIG. 4.Alternatively, fine frit silicon carbide “wet-dry” sandpaper can beused. The silicates in grout or tile can abrade and shape therestrictor. Diamonds are preferred.

Layers of the restrictor may also be etched away chemically. Forexample, selective layers of a glass restrictor may be etched with HF.One procedure that may be used is to force air in the tip slowly and dipthe restrictor deeper and deeper into hydrofluoric acid (aq.). This willtaper the end to a pencil-like tip point. Other suitable chemicals maybe used. For example, if the restrictor is metal, an acid such ashydrochloric acid may be used to remove layers. Combinations ofmechanical and chemical etching may be used.

The angle of the conical tip may be varied from very acute to very sharp(from about 5 degrees from the plane of the body of the restrictor toabout 89 degrees from the plane of the body of the restrictor). Theangle of the conical tip determines the volume in which the dropletswill form, with a more acute angle increasing the volume at which thedroplets will form. When the angle is more acute, the channel outletsappear as long ellipsoids, rather than round holes. Spraying over thearea of a hemisphere (rather than in a single direction) allows dropletsto be rapidly dispersed in the drying gas with less agglomeration.

The benefits of using a multichannel restrictor include increasedthroughput relative to a single channel restrictor and the ability tocontinue forming particles if some channels are blocked or clogged. Thebenefits of using an elongated exit for fluids include: 1) a moredispersed (axially) plume of aerosols which helps avoid problems ofaggregation of particles after formation and 2) a better mixed fluid.

The multichannel restrictor may be used to form particles of varioussubstances, including fluids; melts; solutions; supercritical fluids andsolutions or suspensions of supercritical fluids and aqueous and/ororganic solvents; emulsions; microemulsions; micelles; reverse micellesand other substances into aerosols containing fine particles of solids(amorphous or crystalline). These aerosols may be dried using themethods described herein, or other methods known to the art. Themultichannel restrictor may also be used in fire extinguishers, wherefree radical scavengers can be used in combination with removal of heatto smother a fire. An aerosol cloud of very finely divided waterdroplets containing radical scavenger has higher surface areas anddroplet suspension lifetime without sedimentation than larger waterdroplets prepared by conventional spray nozzles without CD₂.

EXAMPLES Materials

Microcrystalline egg white lysozyme, crystallized (3×), dialyzed inwater and lyophilized was purchased from Sigma Chemical Co. lot#53H7145.Lactate dehydrogenase was obtained as an ammonium salt suspension,purchased from Sigma Chemical Co. (isolated from rabbit muscle, M₄isoenzyme lot#95H9550) and from Boehringer-Mannheim (isolated fromporcine heart, M₄ isoenzyme, Batch #84895527). Sucrose and mannitol werepurchased from Pfanstiehl Laboratories and used without furtherpurification. Polyoxyethylene (20) sorbitan monolaurate (Tween 20) andbuffer salts were purchased from Aldrich Chemical and used withoutfurther purification. Carbon Dioxide (SFE grade, siphon tank) waspurchased from Scott Specialty Gases.

Methods

Preparation of the Enzymes. Lysozyme solutions were prepared by theaddition of the solid, previously lyophilized, material to the desiredformulation. Lysozyme is a fairly robust protein that is notsignificantly damaged upon lyophilization or conventional spray drying.The lyophilized powder was allowed to slowly diffuse into solution at atemperature between 2 and 8° C. The LDH ammonium sulfate suspensionswere dialyzed against 100 mM potassium phosphate (pH 7.5) at atemperature between 2 and 8° C. for 12 to 24 hours. The resultingsolution was then diluted to a concentration of 100 mg/mL of protein inwater.

Nebulization and Bubble Drying System. The system used for supercriticalCO₂-assisted nebulization is summarized here briefly (diagram in FIG.1).An aqueous stream and a stream of supercritical or near critical carbondioxide (T>32° C., P=1500 psi) were each delivered at a constant flowrate of approximately 0.3 mL/min into each of two legs of a low deadvolume mixing tee (Valco) using a HPLC solvent delivery pump (Watersmodel M-6000A) for the aqueous stream and a syringe pump (ISCO Model260D, set to deliver at a constant pressure of 1500 psi) for the carbondioxide. The two streams, initially at room temperature, were heated tojust above 32° C., by a thermocouple-controlled cartridge heaterattached to the mixing tee. The resultant emulsion that formed insidethe mixing tee was allowed to expand out of the third orifice of the teewhich was fitted with a 50 μm inner-diameter 5 cm long fused silicapressure restrictor (Alltech). The rapid decompression of thesupercritical fluid as it exited the pressure restrictor, coupled withthe explosive release of dissolved carbon dioxide from the aqueoussolution caused the formation of very fine aqueous droplets containingsome residual dissolved carbon dioxide. This aerosol was then directedinto a custom-built drying chamber consisting of a 30 cm×2 cmborosilicate glass tube fitted with 4 gas inlet ports at the top of thetube and a powder filtration apparatus (stainless steel Millipore filterholder, 0.2 μm pore size cellulose acetate filter paper) followed by acold trap and vacuum pump at the bottom of the tube. Heated dry nitrogenat a flow rate of approximately 15 L/min was added concurrently with theaerosol through the four gas inlet ports at the top of the drying tube.Additionally, the tube was heated externally with an infrared lamp toaid in the drying process and to keep water from condensing on the innerwalls of the tube. The resultant temperature inside the drying chamberwas maintained below 70° C. during nebulization, which was sufficient tocause rapid bubble drying.

Once the system was equilibrated by nebulizing and drying pure water for10 to 15 minutes, an aqueous protein formulation (containing between 2and 20% w/w total solids) was injected into the aqueous feed line via anHPLC-type injection port. This type of injection port allows for verysmall volume aliquots (between 0.5 and 5 mL) of the aqueous proteinformulation to be nebulized, dried and collected, which is especiallyuseful when working with limited quantities of protein. The design ofthis system allowed for the collection of the dried powder under aconstant purge of warm dry nitrogen, which was continued aftercompletion of the experiment to remove excess water vapor from thedrying chamber. The filter paper apparatus was then isolated from thesystem and transferred to a dry nitrogen-purged glove bag, where thepowder was transferred into 1.5 mL microcentrifuge tubes and capped andstored in desiccators over conc. sulfuric acid until analysis. In someexperiments, the vacuum pump was used to draw the water vapor, carbondioxide, and nitrogen through the filter at pressures slightly belowatmospheric (630 mm Hg in Boulder, Colo. at its mile-high elevation). Inother experiments the vacuum pump was eliminated and the drying wasconducted at pressures slightly above ambient, with equivalent results(data not shown).

Static and Dynamic System pH Measurements

In order to simulate the conditions inside the low dead volume mixingtee in an observable static system, unbuffered and buffered solutionsmixed with Fisher pH indicating solution were filled into a highpressure chamber equipped with sapphire windows, a pressure transducer,thermocouple relay-controlled heating cartridges and a magnetic stirringmechanism. The contents of the cell were equilibrated at 35° C., withstirring, and pressurized to 1500 psi with CO₂. In order to determinethe degree of increased acidity due to pressurization of the aqueousformulations with carbon dioxide, a series of pH measurements was madeon buffered and unbuffered solutions. In a second, more quantitativeapproach, buffered and unbuffered solutions were nebulized with thesupercritical CO₂ system and collected as wet aerosols until the levelof the aqueous solution in the collection vessel permitted a measurementby a pH probe.

Experimental Design

Samples for powder characterization were prepared with supercriticalCO₂-assisted nebulization and bubble drying from the following aqueouslysozyme formulations: “buffer only” consisted of 4 mg/mL lysozyme and100 mM potassium phosphate buffer (pH 7.0). “10% mannitol” consisted of4 mg/mL lysozyme, 100 mM potassium phosphate buffer (pH 7.0) and 10% w/wmannitol. “10% sucrose” consisted of 4 mg/mL lysozyme, 100 mM potassiumphosphate buffer (pH 7.0) and 10% w/w sucrose and “10% sucrose w/Tween”consisted of 4 mg/mL lysozyme, 100 mM potassium phosphate buffer (pH7.0), 10% w/w sucrose and 0.01% w/w Tween 20 (polyoxyethylene (20)sorbitan monolaurate). Prior to nebulization, all formulations werefiltered through 0.2 μm Nylon syringe filters to remove anyparticulates. Approximately 2 mL of each formulation was injected intothe nebulization system for each run, except for the buffer onlyformulation, which was injected at 6 mL volume per run due to the lowtotal solute content of this formulation (in an effort to collect enoughpowder per run for analysis). Each formulation was injected three timesand each powder batch was collected separately to determine the run torun consistency of the system. Additionally, powders containing LDH wereprepared from aqueous formulations consisting of 0.1 mg/mL LDH and 100mM potassium phosphate (pH 7.5), and with the following excipients: 10%w/w mannitol; 10% w/w sucrose; and 10% w/w sucrose with 0.01% w/w Tween20. These concentrations refer to percentages in the aqueous solutionbefore bubble drying; the concentrations in the dried powders becomeproportionately larger as the water is removed.

Size Exclusion Chromatography

The presence of soluble aggregates was determined by size exclusionchromatography performed on a Dionex HPLC equipped with a model AD20 UVdetector set to 280, a GP40 gradient fluid pump and a Spectra Physicsautosampler. The stationary phase consisted of a 30 mm×7.8 mm (i.d.)TosoHaas TSK-Gel G3000SW_(x1) column of 5 mm silica beads with a poresize of 250 Å. The isocratic mobile phase consisted of 100 mM potassiumphosphate buffer (pH 7.0). Prior to injection, powder samples wererehydrated to the original, pre-dehydrated, total solids weight to totalsolution weight ratio with deionized water. The rehydrated samples werecentrifuged and loaded into the autosampler in septa-capped HPLC vials.The flow rate was set to 0.5 mL/min, and approximately 10 mL of eachsample was injected onto the column. Horseradish peroxidase, myoglobin,BSA, and ovalbumin were used as molecular weight standards. Each samplewas injected three times and an average of each peak area wasdetermined.

Scanning Electron Microscopy

Powders were examined using an ISI-SX-30 and a Jeol JSM-6400 scanningelectron microscope (SEM) operating at an acceleration voltage of 30 kV.Samples were adhered to aluminum stubs using carbon tape and were goldsputter coated prior to analysis.

Thermal Analysis

Differential scanning calorimetry (DSC) was performed on a Perkin-ElmerDSC-7. Powders (5 to 10 mg) were loaded into anodized aluminum pans andhermetically sealed under nitrogen in a humidity controlled atmosphere(<2% relative humidity). Each sample was rapidly cooled to −20° C., heldat −20° C. for 10 min and then heated to 200° C. at a heating rate of10° C./min. T_(g) is the glass transition temperature (glass→liquid).T_(m) is the melting temperature of solid→liquid.

X-ray Powder Diffraction

Powder X-ray diffraction was carried out on a Sintag PADV systemequipped with a CuK_(a) source (1=1.54056 Å) operating at a tube load of40 kV and 25 mA. To determine the presence or absence of crystallinityin the powders prepared by supercritical CO₂-assisted nebulization andbubble drying, samples were scanned over a range of 2θfrom 5° to 50° ata scan rate of 0.02°/minute.

Moisture Analysis

Karl Fisher titrations were performed on a Mettler Karl Fisher automatictitrator. Samples for analysis were prepared in a nitrogen purged glovebox maintained below 1% relative humidity. Approximately 1 mL ofanhydrous methanol or formamide was added to 25 mg of sample powder. Theresulting suspension was sonicated for several minutes. 100 mL of thesolution was injected into the coulometer and the moisture content ofthe powder was determined after subtraction of the background moisturecontent of the solvent in which the powders were suspended. Analysis ofeach powder sample was performed in triplicate.

Infrared Spectroscopy

Infrared spectra were obtained on a Bomem PROTA infraredspectrophotometer. Lyophilized powders (ca. 0.5 mg protein) were mixedwith 300 mg anhydrous KBr and compressed into a pellet with a 13 mmevacuable die. The pellets were placed directly into the nitrogen purgedsample chamber of the spectrophotometer. Aqueous solutions of nativeprotein (20 mg/mL) were placed in a sample cell containing CaF₂ windowsseparated with a 6 mm Mylar spacer. An average of 128 scans wascollected with 4 cm⁻¹ resolution in the 4000-900 cm⁻¹ range and Fouriertransformed. For aqueous samples, the spectra of liquid water and watervapor were subtracted from the protein spectra of the protein solutionsaccording to previously established criteria. (Dong, A. et al. (1990),“Protein secondary structures in water from second-derivative amide Iinfrared spectra,” Biochem. 29:3303-3308; Dong, A. and Caughey, W. S.(1994), “Infrared methods for study of hemoglobin reactions andstructures,” Methods Enzymol. 232:139-175; Dong, A. et al. (1995),“Infrared Spectroscopic Studies of Lyophilization—andTemperature—Induced Protein Aggregation,” J. Pharn. Sci. 84:415-424.)The second derivative for the Fourier transformed spectra was calculatedand a Savitzky-Golay smoothing of the data was applied with aseven-point convolution window to reduce possible white noise.

Lysozyme Enzymatic Activity Assay

A bacterial suspension of Micrococcus lysodeikticus (Sigma lot #38H8619)at a concentration of 0.25 mg/mL in 67 mM phosphate buffer (pH 6.6) wasprepared. The lysozyme solutions were diluted to 4 mg/mL enzyme with thephosphate buffer. The reaction mixture consisted of 2.5 mL of the cellsuspension and 0.1 mL of the enzyme dilution. The enzymatic activity wasproportional to the rate of the decrease in turbidity of the cellsuspension, which was measured spectrophotometrically as a lineardecrease in absorbance at 450 nm for 2 min.

LDH Enzymatic Activity Assay

LDH activity was measured at 25° C. in a 2 mL reaction mixtureconsisting of 25 mM tris(hydroxymethyl)aminomethane/tris(hydroxymethyl)aminomethane hydrochloride (Tris/Tris HCl, pH 7.5), 100 mM KCl, 2 mMpyruvate (Sigma, lot #126H10981), and 0.15 mM NADH (Sigma lot#027H78191). The LDH preparation (10 mL) was added to the reactionmixture, the cuvette was inverted 3× and the absorbance decrease at 340nm was monitored with a spectrophotometer. Activity was measuredimmediately prior to supercritical CO₂-assisted nebulization andimmediately after rehydration of the dried powders and reported as apercentage of initial activity. Note in FIG. 11 the LDH activity isgreater than 100% of the activity of the initial aqueous solution forcertain compositions.

Static and Dynamic pH Measurements with and without Buffers

The question of whether the acidification occurring when CO₂ isdissolved in water could be avoided by the use of buffers was studied.The solubility of CO₂ (approximately 2 mole % at 35° C.) in water at1500 psi causes an increase in acidity in unbuffered solutions. In theabsence of buffers, an acidic environment could potentially denatureproteins. For these reasons, a static system was used to simulate theconditions within the mixing tee in order to observe the extent of theincrease in acidity of an aqueous solution without buffering. pHsensitive indicator dyes were added to the aqueous solution prior topressurization with carbon dioxide to qualitatively monitor the changein acidity. As expected, the pH rapidly decreased below the indicatorrange (pH<4) immediately upon pressurization. The capability toneutralize this effect was estimated with this static system for thefollowing buffer salts: monobasic/dibasic potassium phosphate (pH 7),Tris/Tris HCl (pH 7.2), citric acid/sodium citrate (pH 5.5), and aceticacid/sodium acetate (pH 5). The buffering capacity for all solutionstested, measured as the ability of the buffer system to forestall thecolor change of the indicator dye, leveled off at approximately 100 mMconcentration (data not shown). To further quantitate theseobservations, the same buffers used in the static system were nebulizedwith the supercritical CO₂ system and collected as wet aerosols. The pHof each solution had to be measured immediately after collection as thevalue continued to increase due to carbon dioxide effervescence. Theresults of this experiment are presented in FIG. 5 and are consistentwith the static observations that 100 mM buffer will adequatelyneutralize the effects of dissolved CO₂ and avoid damage to proteinsthat are damaged by acidification.

Fine Powder Characterization

In order to determine the surface characteristics of molecules producedby the method of the current invention, fine powders of albuterolsulfate, tobramycin sulfate, cromolyn sodium, rhDNase, lactose, andsodium chloride were prepared using the method outlined above.Conditions for droplet formation were CO₂ pressure: 1500 psi, 0.3 mL/minflow rates, CO₂ and H₂O concentrations were about 10% (wt/vol), tubeinlet ˜70° C., tube outlet ˜50° C., 50 mm, 5 cm fused silica restrictor,0.2 mm cellulose acetate filter paper.

FIG. 6 shows electron micrographs of representative dry powders preparedusing the method of this invention. FIG. 6A is a transmission electronmicroscopy (TEM) image of dried sodium chloride particles prepared fromthe method of the invention using a 20% aqueous sodium chloride sample.FIG. 6B is a scanning electron microscopy (SEM) image of the same sodiumchloride solution after drying by the method of this invention. FIG. 6Cis a TEM image of mannitol. FIGS. 6D and 6E show tobramycin sulfateparticles (6D) and 1% tobramycin sulfate in lactose particles (6E)prepared by the method of this invention on a 10% (total solutemass/volume) aqueous solution. FIG. 6F is an image of albuterol sulfateparticles produced by the method of this invention. FIG. 6G is an imageof cromolyn sodium particles produced by the method of the invention.

Dry powders were initially prepared by CO₂-assisted nebulization andbubble drying from the aqueous lysozyme formulations described inMaterials and Methods. Visually, the collected samples were all veryfine white powders with very little run to run variability in thequality of the powders. All powders were hygroscopic in moist air, butwere free flowing and easily handled in a low-moisture environment.Electron micrographs of representative dry powder protein formulationare shown in FIG. 7. “Buffer only” powder (A) 10% mannitol (B), 10%sucrose (C), and 10% sucrose with 0.01% Tween 20 (D). Note that whenTween is used, (see FIG. 6D), a powder with less agglomerated particlesand more distinctly spherical, individual particles is formed. This maybe a direct or indirect effect from the addition of surfactant Tween 20.Without wishing to be bound by any particular theory, it is believedthat the detergent aids in dehydration during processing. All powdersproduced had significant portion of particles well within the 1 to 3 mmdiameter inhalable size range. The morphology of the powders preparedfrom the buffer only formulation and the formulation containing 10%mannitol appear to be significantly less spherical and smooth than thepowders prepared from the formulations containing sucrose. The additionof Tween 20 to the sucrose solution had an effect to even further smoothout the surface of the dried particles. This has been observed by otherresearchers for particles that were conventionally spray-dried and it ispostulated that the addition of a surface active agent to the aqueousformulation has an effect to reduce surface turbulence during thedehydration process.

The micrograph of the 10% mannitol formulation (FIG. 7B), indicates amore crystalline material as seen by the multi-faceted particles.

Moisture Analysis

Karl Fischer moisture analysis on lysozyme powders dried by the methodof the invention also indicated low moisture contents: the moisturecontent of samples of 4 mg/mL lysozyme with 10% (percentage in initialaqueous solution) mannitol was 0.6±0.05%; the moisture content ofpowders of 4 mg/mL lysozyme with 10% sucrose was 1.18±0.02% and themoisture content of powders of 4 mg/mL lysozyme with 10% sucrose and0.01% Tween was 1.25±0.07%.

The Karl Fisher titration results performed on each LDH powder preparedby supercritical CO₂-assisted nebulization and bubble drying indicatethat quite low moisture contents are attainable for this new process:the moisture content of the powder prepared from the buffer only aqueousformulation was 0.83+0.06% w/w, 1.3+0.01%, from the 10% mannitolformulation, 1.4+0.08% form the 10% sucrose formulation, and 3.25+0.03%from the formulation containing 10% sucrose and 0.01% Tween 20. Thecollected sucrose powders were very hygroscopic, and had to be handledin a gloved bag under an atmosphere of dry nitrogen. Other sugarpowders, e.g., trehalose, are less hygroscopic.

XRD and DSC

Because the crystallization of excipients can have dramatic effects onthe solubility and stability of solid pharmaceutical formulations, theability to form crystalline and/or amorphous powders with supercriticalCO₂ assisted nebulization was investigated. The strong tendency ofmannitol to crystallize during freeze-drying and to form crystallinepowders upon spray drying is well known. Therefore, mannitol was chosenas an excipient to determine the capability of the nebulization systemto form crystalline powders, although this excipient was not expected tostabilize the model proteins to a significant degree. Whilecrystallinity is a desired attribute for excipients used as bulkingagents for solid pharmaceuticals, excipients that tend to crystallizeduring processing offer very little protection to labile protein drugsduring dehydration and subsequent storage in the dried state. The powderdiffraction data for a lysozyme sample prepared from the 10% mannitolformulation appears in FIG. 8 for samples prepared by supercriticalCO₂-assisted nebulization. Aqueous formulations contained 4 mg/mLlysozyme, 100 mM phosphate buffer at pH 7.0 and were nebulized togetherwith 10% mannitol (A) or 10% sucrose (B). Final approximate solid weightpercentage in the bubble dried powders: 4% protein, 16% buffer and 80%sugar. The diffraction data of the powder prepared from the 10% mannitolsolution indicates good crystallinity within the sample and appears tobe a mixture of the a and b polymorphs of mannitol. This diffractionpattern is consistent with the powder diffraction pattern obtained forpure D-mannitol as received from the vendor (not shown).

DSCs for mannitol as received, mannitol spray dried with lysozyme,sucrose spray dried with lysozyme and sucrose and lysozyme are notshown. The DSC of a sample of mannitol as received showed a peak from157.25 to 177.56° C.; onset 156.10° C. T_(m) was 159° C.; 300.47 J/g.PSC of a sample of mannitol spray dried with 4 mg/mL lysozyme showed apeak onset from 77.63 to 146.48° C. T_(m)=154.7° C.; 134 J/guncorrected. DSC of sample of sucrose with 4 mg/mL lysozyme showed T_(g)from 21.93 to 47.12° C. with onset 40.25° C. T_(g)=43.05° C. and 1.51J/g deg uncorrected. DSC of sucrose with Tween and 4 mg mL lysozymeshowed T_(g) from 28.57 to 56.99° C.; onset 45.36° C.; 1.00 J/g deg;T_(g)=50.95° C.

The X-ray powder diffraction data of the sucrose formulations (FIG. 6B),indicate a more amorphous nature confirmed by the glass transitionobserved in the DSC thermograms. The ability to form amorphous glassypowders below the glass transition temperature is an importantcapability of the disclosed system, as significant evidence has beenfound recently linking protein stabilization with vitrification (glassformation) (Crowe, J. H. et al. (1998), “The Role of Vitrification inAnhydrobiosis,” Annu. Rev. Physiol. 60:73). Differential scanningcalorimetry of the sample indicated the presence of an endothermic eventat 154.7° C., which is consistent with the melting temperature ofcrystalline D-mannitol (169° C.). The depressed T_(m) is probably due tothe presence of water (approximately 1% as measured by Karl Fishertitration) in the powder.

Characterization of Rehydrated Solutions

Powders collected on the filter paper were transferred to small inerttubes and rehydrated with distilled, deionized water to the originalwt/wt% (total solute mass/total solution mass). Activity assay of therehydrated protein solutions compared to the original solutionsindicated greater than 90% activity retention (or recovery) for allsolutions sprayed. HPLC (size exclusion Tosohaas column TSK3000SW_(XL),100 mM KPO₄ pH=7.0 elution buffer) indicated that the ratio of monomericprotein to dimeric aggregates remained constant after drying andrehydration (aggregate formation to produce dimers, trimers andeventually insoluble aggregates is a common protein degradationpathway). HPLC data are not shown. The aggregate:monomer ratio wascalculated to be approximately 0.21%±0.02% for the initial lysozymeformulations and for all rehydrated powders.

Researchers believe that one important method for stabilizing a proteinin the solid state is to provide the protein with a matrix in whichnative protein structural conformation can be, at least somewhat,preserved upon dehydration (Chang, B. S. et al. (1996), “PhysicalFactors Affecting the Storage Stability of Freeze-Dried Interleukin-1Receptor Antagonist: Glass Transition and Protein Conformation,” Arch.Biochem. Biophys. 331:249; Allison, D. S. et al. (1998), “Effects ofDrying Methods and Additives on Structure and Function of Actin:Mechanisms of Dehydration-Induced Damage and Its Inhibition,” Arch.Biochem. Biophys. 358:171). Dehydration-induced structural transitionshave been shown to be inhibited by the addition of certain stabilizers,such as sucrose, to the protein solution before processing (Prestrelski,S. J. et al. (1993), “Dehydration-induced Conformational Transitions inProteins and Their Inhibition by Stabilizers,” Biophys. J. 65:661).Second derivative Fourier-transformed infrared spectroscopy (Carpenter,J. F. et al. (1998), “Application of Infrared Spectroscopy toDevelopment of Stable Lyophilized Protein Formulations,” Eur. J. Pharm.Biopharm. 45:231) in the amide I region (1600-1700 cm⁻¹) was used toprobe the conformational structure of the dried lysozyme powders.Representative results are shown in FIG. 9. Samples of (A) lysozyme inthe native aqueous state; (B) lysozyme containing 10% sucrose and 0.01%Tween 20; (C) lysozyme with 10% sucrose; (D) lysozyme with 10% mannitol;and (E) lysozyme with buffer only were dried at 70° C. The amide Iregion shows characteristic C=0 stretching frequencies for differenttypes of secondary structures. Interestingly, while enzymatic activitywas almost fully retained upon rehydration of all powders, the driedlysozyme powders showed markedly different protein conformations in eachformulation. When compared to the native lysozyme liquid infraredspectrum, the formulation without any excipient showed the largestdeviation from native conformation, the 10% mannitol formulation showedsomewhat less deviation from the buffer only formulation, while the 10%sucrose formulation showed even less deviation from the nativestructure. The 10% sucrose plus Tween 20 formulation showed the mostnative-like structure of all the powders. These results indicate thatlysozyme was not irreversibly damaged by our nebulization and dryingprocess. The protein structure, however, showed significant deviationfrom the native conformation in the dry powders. The recovery ofactivity seems to indicate the ability of lysozyme to recover its nativeconformation upon rehydration. The conformational transitions observedin the drying process can be inhibited by the addition of disaccharidestabilizers such as sucrose.

Without wishing to be bound by theory, it is believed that sugarshydrogen bond to the protein in place of water molecules, therebyreducing conformational changes in the protein structure upondehydration. It is believed that surfactants reduce the amount of stressat the air-water interface that the protein is subjected to by competingfor the available air-water interface regions with the protein.Surfactants added to the protein formulation are believed to preventaggregation of the protein molecules.

Lysozyme Activity Assay

Enzymatic activity of the lysozyme powders rehydrated to their originaltotal solute % (w/w) was measured immediately after rehydration. Theseresults (FIG.10) are reported as a percentage of the initial activitythat was independently measured immediately before supercriticalCO₂-assisted nebulization and bubble drying. Results are an average ofat least three measurements, and error bars represent one standarddeviation. Analysis of lysozyme powders indicated greater than 90%recovery of initial activity for all formulations. The observed recoveryof activity is an indication of the reversibility of the structuralchange in this protein upon dehydration and rehydration. Thus, althoughlysozyme is severely unfolded upon dehydration during supercriticalCO₂-assisted nebulization in the absence of amorphous stabilizers, itcan readily refold upon rehydration, and, thereby, recover much of itsoriginal biological activity.

LDH Activity Assay

In order to further examine the nebulization process, studies wereperformed using a significantly more labile enzyme, lactatedehydrogenase (LDH, Sigma, L-5762). Data are shown in FIGS. 10 and 11.LDH is known to be damaged by conventional drying processes (Adler, M.and Lee, G. (1999), “Stability and Surface Activity of LactateDehydrogenase in Spray-Dried Trehalose,” J. Pharm. Sci. 88:199). LDH wasdialyzed against the 100 mM potassium phosphate buffered to pH=7.5 atroom temperature for at least 12 hours. Protein concentration was keptconstant at 100 μg/mL. The LDH formulations were dehydrated using thesame conditions used for bubble drying the lysozyme formulations. Thepowders prepared from the LDH solutions appeared visually identical tothe powders containing lysozyme (data not shown).

Dry powders comprising LDH were produced from formulations containing10% mannitol, 10% sucrose and 10% sucrose plus 0.01% Tween 20, alongwith LDH (100 μg/mL) and potassium phosphate buffer (100 mM, pH=7.5).The activity of the rehydrated powders was compared to the originalformulations before nebulization and drying.

Four different formulations were prepared:

Buffer only

0.1 mg/mL LDH

100 mM KPO₄ (pH 7.5)

10% Mannitol

0.1 mg/mL LDH

100 mM KPO₄ (pH 7.5)

100 mg/mL mannitol

10% Sucrose

0.1 mg/mL LDH

100 MM KPO₄ (pH 7.5)

100 mg/mL sucrose

10% Sucrose with Tween

0.1 mg/mL LDH

100 mM KPO₄ (pH 7.5)

100 mg/mL sucrose

0.1 mg/mL Tween 20

The LDH catalytic activity recovery for these formulations is shown inFIG.11. LDH, nebulized and dehydrated without any added excipients,recovers only 15% of its original activity after rehydration.Interestingly, the same solution collected as a wet aerosol retained 87%of its original enzymatic activity. This result indicates that most ofthe damage experienced by the protein occurs in the drying process afterthe aqueous solution/CO₂ emulsion is ejected from the restrictor tip. Inother words, CO₂-assisted nebulization is not as harsh as the drying ofmicro-droplets and micro-bubbles in nitrogen at 70° C. The irrecoverabledamage experienced during the dehydration process was partiallyprevented by the addition of mannitol, and to a greater degree withsucrose. Activity loss was almost completely prevented by the additionof sucrose and Tween 20. These results are as expected from thestructural analysis of the lysozyme formulations as LDH, a more labile,tetrameric enzyme is not expected to have the same ability to refoldupon rehydration as lysozyme. As a result, activity was better preservedwhen LDH was dried in the presence of sucrose as it is most likely thatthe protein's structure was preserved.

FIG. 12 compares recovery of activity upon rehydration for varyingpercentages of mannitol, sucrose and (sucrose+Tween (0.01%)). Note thatthe percent recovery of initial activity shows the most dramatic changewith concentration of sugar when Tween is used in the formulation.

In general these results indicate that the damage experienced by LDHduring the supercritical CO₂-assisted nebulization and bubble drying canbe inhibited by the addition of a sugar stabilizer and even further withthe combination of 10% sucrose and 0.01% surfactant, Tween 20. Theformulated protein appears to be successfully protected throughout thedehydration process experienced during supercritical CO₂-assistednebulization and bubble drying.

The mechanism(s) by which Tween 20 increased protection in the presenceof sucrose during supercritical CO₂-assisted nebulization and bubbledrying is uncertain at this point. Tween 20 may protect the protein bysaturating surface sites at the air-liquid interface thereby preventingsurface denaturation. The air-liquid interface is expected to besignificant for this process due to the high surface area micro-dropletsproduced by supercritical CO₂-assisted nebulization. Additionally, thesurfactant may offer protection by saturating hydrophobic sites on thesurface of the protein, which are potential sites for aggregation.Furthermore the surfactant may foster protein refolding duringrehydration. Whether Tween 20 is protecting LDH by one or more of thesemechanisms during nebulization and bubble drying is undetermined at thispoint. This protein protection effect, combined with the improvedparticle separation and smoothness noted in sucrose formulationscontaining Tween 20, clearly document the potential benefits ofincluding this surfactant in formulations dried by supercriticalCO₂-assisted nebulization and bubble drying. It was also noted in someC., experiments that the enzymatic activity of aqueous LDH aftercompression with CO₂, decompression, and bubble drying was apparentlygreater, as indicated by enzymatic analysis, than the initial aqueoussolution before compression at 2000 psi (see FIG.12). It has beendemonstrated by St. John, Carpenter and Randolph that protein refoldingis facilitated by pressurizing aqueous solutions at 20,000 psi (“HighPressure Fosters Protein Refolding from Aggregates at HighConcentrations,” Proc. Natl. Acad. Sci. 96:13029, 1999). At this time,the cause of the improved activity is not known. Although Applicant doesnot wish to be bound by theory, treating aqueous solutions bycompression with CO₂ with a surfactant may produce apparent improvedactivity due to the surfactant, the moderate pressure, the presence ofcarbon dioxide, or sugar, or by other mechanisms.

Although the description above contains many specificities, these shouldnot be construed as limiting the scope of the invention, but as merelyproviding illustrations of some of the presently-preferred embodimentsof this invention. For example, many fine dry powders of many differentsubstances can be used in the methods of the invention; othersupercritical or near-critical fluids may be used than specificallyexemplified; and additives other than those specifically exemplified maybe used. The scope of the invention should be determined by the appendedclaims and their legal equivalents rather than by the examples given.

Each reference cited in the present disclosure is incorporated byreference herein to the extent not inconsistent with the disclosureherein.

We claim:
 1. A method of forming fine dry particles, wherein theparticles comprise substances which are either soluble in supercriticalfluid, near critical fluid, or mixtures thereof, or substances which aresoluble or suspendable in aqueous solutions, which method comprises: (a)forming a composition comprising one or more substances and asupercritical or near critical fluid; (b) reducing the pressure on saidcomposition, whereby droplets are formed; (c) passing said dropletsthrough a flow of drying gas which is not the same substance as thesupercritical or near critical fluid, said drying gas heated from aboveambient temperature to about 100° C.
 2. The method of claim 1, whereinthe temperature of said drying gas at the point where said droplets areinitially passed through said gas is below about 100° C.
 3. The methodof claim 1, wherein said one or more substances of step (a) is solublein said supercritical or near critical fluid.
 4. The method of claim 1,wherein said composition of step (a) also comprises an aqueous solvent.5. The method of claim 4, wherein said one or more substances of step(a) is soluble in said aqueous solvent.
 6. The method of claim 1,wherein said composition of step (a) also comprises one or moreadditives selected from the group consisting of: excipients,stabilizers, bulking agents and surfactants.
 7. The method of claim 6,wherein said one or more additives comprise less than about 99.9% of theweight of the dry particles.
 8. The method of claim 1, wherein saidcomposition of step (a) also comprises a pH buffering substance.
 9. Themethod of claim 1, wherein said supercritical or near critical fluid iscarbon dioxide.
 10. The method of claim 1, wherein said drying gas isnitrogen.
 11. The method of claim 1, wherein said flow of drying gas ofstep (c) is contained within a drying chamber.
 12. The method of claim1, wherein said one or more substances of step (a) comprise aphysiologically active composition selected from the group consisting ofsurfactants, insulin, amino acids, enzymes, analgesics, anti-canceragents, antimicrobial agents, viruses, antiviral agents, antifungalpharmaceuticals, antibiotics, nucleotides, DNAs, antisense cDNAs, RNAs,peptides, proteins, immune suppressants, thrombolytics, anticoagulants,central nervous system stimulants, decongestants, diureticvasodialators, antipsychotics, neurotransmitters, sedatives, hormones,anesthetics, anti-inflammatories, antioxidants, antihistamines,vitamins, minerals and other physiologically active materials known tothe art.
 13. The method of claim 1, further comprising: (d) collectingsaid fine dry particles.
 14. A method of forming fine dry particlescomprising: (a) mixing an aqueous solution containing one or moresubstances of interest and a supercritical or near supercritical fluid,forming a composition; (b) reducing the pressure on said composition,whereby droplets are formed; (c) passing said droplets through a flow ofdrying gas which is not the same substance as the supercritical or nearcritical fluid, said drying gas heated from above ambient temperature toabout 100° C.
 15. The method of claim 14, wherein the temperature ofsaid drying gas of step (c) at the point where said droplets areinitially passed through said drying gas is below about 100° C.
 16. Themethod of claim 14, wherein said composition of step (a) also comprisesone or more additives selected from the group consisting of: excipients,stabilizers, bulking agents and surfactants.
 17. The method of claim 16,wherein said one or more additives comprise less than about 99.9% of theweight of the dry particles.
 18. The method of claim 16, wherein ifpresent, surfactants are present at a concentration of between about0.001 to 0.5 wt %; and if present, stabilizers are present at aconcentration of between about 0.05 to 25 wt %.
 19. The method of claim14, wherein the mixing is performed in a low dead volume tee.
 20. Themethod of claim 14, wherein said one or more substances of step (a)comprise a physiologically active composition selected from the groupconsisting of surfactants, insulin, amino acids, enzymes, analgesics,anti-cancer agents, antimicrobial agents, viruses, antiviral agents,antifungal pharmaceuticals, antibiotics, nucleotides, DNAs, antisensecDNAs, RNAs, peptides, proteins, immune suppressants, thrombolytics,anticoagulants, central nervous system stimulants, decongestants,diuretic vasodialators, antipsychotics, neurotransmitters, sedatives,hormones, anesthetics, anti-inflammatories, antioxidants,antihistamines, vitamins, minerals and other physiologically activematerials known to the art.
 21. The method of claim 14, wherein saidflow of drying gas of step (c) is contained within a drying chamber. 22.A method of forming fine dry particles comprising: (a) equilibrating anaqueous solution of a substance of interest with a supercritical or nearsupercritical fluid, forming a composition; (b) reducing the pressure onsaid composition, whereby droplets are formed; (c) passing said dropletsthrough a flow of drying gas which is not the same substance as thesupercritical or near critical fluid, said drying gas heated from aboveambient temperature to about 100° C.
 23. The method of claim 22, whereinthe temperature of said drying gas of step (c) at the point where saiddroplets are initially passed through said drying gas is below about100° C.
 24. The method of claim 22, wherein said composition of step (a)also comprises one or more additives selected from the group consistingof: excipients, stabilizers, bulking agents and surfactants.
 25. Themethod of claim 24, wherein saidone or more additives comprise less thanabout 99.9% of the weight of the dry particles.
 26. The method of claim24, wherein if present, surfactants are present at a concentration ofbetween about 0.001 to 0.5 wt %; and if present, stabilizers are presentat a concentration of between about 0.05 to 25 wt %.
 27. The method ofclaim 22, wherein said substance of step (a) is a physiologically activecomposition selected from the group consisting of surfactants, insulin,amino acids, enzymes, analgesics, anti-cancer agents, antimicrobialagents, viruses, antiviral agents, antifungal pharmaceuticals,antibiotics, nucleotides, DNAs, antisense cDNAs, RNAs, peptides,proteins, immune suppressants, thrombolytics, anticoagulants, centralnervous system stimulants, decongestants, diuretic vasodialators,antipsychotics, neurotransmitters, sedatives, hormones, anesthetics,anti-inflammatories, antioxidants, antihistamines, vitamins, mineralsand other physiologically active materials known to the art.
 28. Themethod of claim 22, wherein said flow of drying gas of step (c) iscontained within a drying chamber.
 29. A device for forming fine dryparticles, wherein the particles comprise a substance or substanceswhich are either soluble in supercritical fluid, near critical fluid, ormixtures thereof, or substance or substances which are soluble orsuspendable in aqueous solutions, consisting essentially of: (a) a firstpressurized chamber containing a first nongaseous supercritical or nearcritical fluid; (b) a second chamber containing the solution orsuspension of the substance or substances in a second nongaseous fluid;(c) a mixing chamber for mixing the solution or suspension of step (b)and first fluid connected to the first and second chambers by conduits;(d) first flow control means connected to the conduit between the firstchamber and the mixing chamber for passing the first fluid into saidmixing chamber; (e) second flow control means connected to the conduitbetween the second chamber and the mixing chamber for passing the secondfluid into said mixing chamber; (f) a restrictor connected to saidmixing chamber for conducting the composition out of the mixing chamberinto an expansion region having a pressure below that of thesupercritical or near critical fluid where a dispersion of fineparticles of said substance or substances is formed; (g) a dryingchamber connected to the restrictor; (h) a source of gas which is notthe same substance as the supercritical or near critical fluid, saidsource of gas connected to the drying chamber at one or more inlets; (i)means for collecting particles after they pass through the dryingchamber.
 30. The device of claim 29, wherein said mixing chamber of step(c) is a low dead volume chamber.
 31. The device of claim 29, whereinsaid first fluid is supercritical carbon dioxide.
 32. The device ofclaim 29, wherein the first fluid of step (a) is a near-critical fluid.33. The device of claim 29, wherein the second fluid of step (b) isaqueous.
 34. The device of claim 29 wherein said substance or substancesis a physiologically active composition selected from the groupconsisting of surfactants, insulin, amino acids, enzymes, analgesics,anti-cancer agents, antimicrobial agents, viruses, antiviral agents,antifungal pharmaceuticals, antibiotics, nucleotides, DNAs, antisensecDNAs, RNAs, peptides, proteins, immune suppressants, thrombolytics,anticoagulants, central nervous system stimulants, decongestants,diuretic vasodialators, antipsychotics, neurotransmitters, sedatives,hormones, anesthetics, anti-inflammatories, antioxidants,antihistamines, vitamins, minerals and other physiologically activematerials known to the art.
 35. A multichannel flow/pressure restrictorfor the formation of particles of a substance from a compositioncomprising a supercritical or near critical fluid, the substance, andoptionally an aqueous or organic solvent or combination of solvents,comprising: (a) an inlet end for the introduction of the composition;(b) an outlet end having plurality of openings; and (c) independentsubstantially parallel non-concentric path channels.
 36. Themultichannel restrictor of claim 35, wherein the outlet end isconically-shaped.
 37. The multichannel restrictor of claim 35 whereinthe channels consist of a honeycomb of ceramic channels with innerdiameters of about 40-50 μm.
 38. The multichannel restrictor of claim 35wherein the restrictor is epoxied to a mixing tee.
 39. A method ofmaking the multichannel restrictor of claim 36 comprising mechanicallyor chemically shaping the outlet end.
 40. The method of claim 39,wherein the mechanical shaping comprises applying stroking pressure tothe outlet end with an abrasive.
 41. The method of claim 39, wherein thechemical shaping comprises etching the outlet end in acid.
 42. A methodof preparing fine dry particles of lactate dehydrogenase (LDH) havingimproved activity upon rehydration than undried aqueous solutions of LDHcomprising: (a) forming a composition comprising less than 1 mg/ml LDH;greater than 5 wt % sugar; less than 0.5 wt % surfactant; water; bufferand a supercritical or near critical fluid where the percentages ofsugar and surfactant are as present in the aqueous solution beforedrying; (b) reducing the pressure on said composition, whereby dropletsare formed; (c) passing said droplets through a flow of gas which is notthe same substance as the supercritical or near critical fluid, saiddrying gas heated to about 70° C.
 43. A method of preparing fine dryparticles of one or more biologically active substances having improvedactivity upon rehydration than undried aqueous solutions of one or moresubstances alone comprising: (a) forming a composition comprising thesubstance or substances; between about 0.05 to 25 wt % sugar; water anda supercritical or near critical fluid; (b) reducing the pressure onsaid composition, whereby droplets are formed; (c) passing said dropletsthrough a flow of drying gas which is not the same substance as thesupercritical or near critical fluid, said drying gas heated from aboveambient temperature to about 100° C.
 44. A device for expansion of acomposition comprising: (a) a low dead volume tee through which saidcomposition passes; (b) a restrictor with more than one substantiallyparallel non-concentric channel affixed to said tee.